The authors from the United States and the Netherlands in their previous work showed in vitro susceptibility and permissivity of dopaminergic (DA) neurons derived from human pluripotent stem cells (hPSCs) to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By contrast, cortical neurons derived from hPSCs did not show susceptibility to SARS-CoV-2. In this article, the same research team investigated molecular changes in dopaminergic neurons in vitro infected with SARS-CoV-2. In addition, to explore whether the changes in DA neurons observed in vitro also occur in vivo, they examined the substantia nigra autopsy samples from patients who died from COVID-19.
Dopaminergic neurons
SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus. Its genome encodes four structural proteins: the spike (S), envelope (E), nucleocapsid (N), and membrane (M) proteins. The S protein is a membrane-bound glycoprotein that binds to the membrane-bound angiotensin-converting enzyme 2 (ACE2) in host cells through the receptor binding domain located in the S1 subunit.
Parkinson’s disease is the second most common neurodegenerative disease, caused by a reduction in the dopaminergic neurons of the substantia nigra, followed by striatal dopamine depletion. Parkinson’s disease is one of the α-synucleinopathies characterized by the misfolding of alpha-synuclein (α-Syn) into pathological forms, resulting in neurodegeneration. SNCA, the first gene associated with familial Parkinson’s disease, encodes the protein alpha-synuclein. Parkinson’s disease is characterized by typical motor symptoms (bradykinesia, rest tremor, and muscle rigidity) and non-motor symptoms (hyposmia, depression, apathy, sleep disorders, and dysautonomia). Viral infections receive more attention as a cause of viral-induced clinical parkinsonism, but the specific mechanism of nigrostriatal dopaminergic neuron degradation after viral infection remains unknown. It is worth noting that a study that employed a computational methodology to predict interactions between human and SARS-CoV-2 proteins revealed that SARS-CoV-2 proteins mimic 43 proteins linked to Parkinson’s disease pathways, including α-Syn. SARS-COV-2 proteins that possibly interact with α-Syn are NSP7 and 3-CL-like (main) protease. (Yapici-Eser H et al. Neuropsychiatric Symptoms of COVID-19 Explained by SARS-CoV-2 Proteins’ Mimicry of Human Protein Interactions. Front. Hum. Neurosci, 23 March 2021) https://www.frontiersin.org/journals/human-neuroscience/articles/10.3389/fnhum.2021.656313/full
About the Study and Results
DA neuron identity was differentiated from hPSCs by immunofluorescence staining with postmitotic DA neuron markers (NURR1: GFP), tyrosine-hydroxylase (TH), MAP2, and FOXA2. Clustering analysis identified four cell populations: three clusters that highly expressed LMO3, a marker of A9 DA neurons, and one cluster that highly expressed CALB1, a marker of A10 DA neurons. These results showed that the population of DA neurons derived from hPSCs was mainly composed of A9 DA neurons (a subtype of DA neurons in the substantia nigra, which is mostly affected by Parkinson’s disease).
At 24, 48, and 72 hours after SARS-CoV-2 inoculation, the infection of purified DA neurons derived from hPSCs was assessed by quantitative real-time PCR (rt-qPCR) analysis. Importantly, the SARS-CoV-2 antigens, including S, E, N, and M proteins, were highly detected in three clusters that expressed a high level of LMO3, a marker of A9 DA neurons, which are mostly affected by Parkinson’s disease in the substantia nigra. In contrast, in a cluster that highly expressed CALB1, a marker of A10 DA neurons, the SARS-CoV-2 antigens were not detected.
The SARS-CoV-2 infection decreased the expression of midbrain DA neuron markers NR4A2, FOXA2, and LMX1A, and in particular, A9 markers LMO3 and DKK3. Quantitative RNA in situ hybridization confirmed the loss of A9 DA neurons. According to these results, the A9 DA neuron subtype, which is mostly affected by Parkinson’s disease in the substantia nigra, was particularly vulnerable to SARS-CoV-2.
The ACE2 blocking prevented SARS-CoV-2 infection of DA neurons derived from hPSCs, suggesting that SARS-CoV-2 infection of DA neurons depends on ACE2 receptors.
Gene analysis revealed that the top-upregulated pathways in SARS-CoV-2-infected DA neurons were the cell cycle, DNA replication, chemokine/cytokine transcripts, inflammation, and senescence pathways. Further analysis confirmed that SARS-CoV-2 triggered cellular senescence in DA neurons, and upregulated senescence-associated genes, including CCL2, CCL20, CSF1, CXCL11, GDF15, IGF2R, IL1B, IL6ST, IQGAP1, and TNFRSF11B. SARS-CoV-2 also upregulated other senescence-associated markers in DA neurons, such as lysosomal senescence-associated β-galactosidase (SA-β-gal) and lipofuscin. DA neurons infected with SARS-CoV-2 showed senescence-associated phenotypes, including increased lysosome accumulation, mitochondrial dysfunction, and protein oxidation.
By contrast, senescence-associated genes were not upregulated in cortical neurons derived from hPSCs, which is consistent with previous data showing that cortical neurons are not susceptible to SARS-CoV-2 infection. Importantly, the senescence pathway was not upregulated in lung organoids, pancreatic cells, liver organoids, and cardiomyocytes infected with SARS-CoV-2.
It is worth noting that the FDA Food and Drug Administration (FDA)-approved drugs riluzole, metformin, and imatinib were found to block SARS-CoV-2-mediated DA neuron senescence. Riluzole, metformin, and imatinib were found to reduce β-gal activity in a dose-dependent manner without cytotoxicity, down-regulate the genes involved in the senescence pathway, and reduce viral RNA in DA neurons infected with SARS-CoV-2.Â
Finally, the authors examined the substantia nigra autopsy samples from six patients who died from COVID-19 and three age-matched controls to determine whether the selective vulnerability of hPSC-derived DA neurons found in vitro was reflected in the brains of COVID-19 patients. The results showed the SARS-CoV-2 transcripts in all six substantia nigra samples. Remarkably, the same transcriptional signatures identified in DA neurons infected with SARS-CoV-2 in vitro were found in autopsy samples, including the induction of chemokines/cytokines, inflammation, and senescence-associated genes. Low viral RNA levels were identified in frozen tissue samples from other brain regions.
Conclusion
This in vitro study demonstrated the selective vulnerability of the A9 DA neuron subtype derived from hPSCs (A9 DA neurons are mostly affected by Parkinson’s disease in the substantia nigra) to SARS-CoV-2 infection and the associated inflammatory and cellular senescence responses. The comparable inflammatory and senescence signatures in the substantia nigra autopsy samples suggest that these results may have clinical relevance. However, the authors emphasized the possibility that other cell types, such as astrocytes or microglia, or other pathological changes, such as a hypoxic state, could contribute to the inflammatory and senescence signatures in the substantia nigra autopsy samples. They recommended that COVID-19 patients should be closely monitored for an increased risk of developing Parkinson’s disease-related symptoms.
This article was published in Cell Stem Cell.
Journal Reference
Yang, L., Kim TW, Han Y et al. SARS-CoV-2 infection causes dopaminergic neuron senescence. Cell Stem Cell 2024; 31, 1–16. https://www.cell.com/cell-stem-cell/fulltext/S1934-5909(23)00442-3
